Optical System and Method for Optically Analyzing Light from a Sample

ABSTRACT

An optical system for analyzing light from a plurality of samples is provided. The optical system includes a plurality of holders adapted to have samples located therein, a collection lens, a transmission grating, and a reimaging lens. The collection lens is configured to receive and substantially collimate light from the samples. The transmission grating is configured to spectrally disperse the substantially collimated light from the collection lens. The reimaging lens is configured to receive the light from the light dispersing element and direct the light onto a light detection device. A method of optically analyzing at least one sample is also provided.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a continuation of U.S. patent application Ser. No.11/193,853 filed Jul. 29, 2005 and a continuation of Ser. No. 10/773,712filed Feb. 5, 2004, now U.S. Pat. No. 6,927,852, which is a continuationof Ser. No. 09/564,790 filed May 5, 2000, now U.S. Pat. No. 6,690,467B1, all of which are incorporated herein by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to an optical system for analyzing light from asample holder. The optical system is particularly suitable for use in asingle or multi-channel separation system. The present invention is alsodirected toward a method of optically analyzing light from a separationsystem using a spectrograph.

2. Background

Spectrographs are devices for separating electromagnetic radiation intoits spectral components. Optical spectrographs can be used for analysisof samples, such as analyzing the chemical composition of nucleic acidsamples in order to determine the nucleotide sequence of the sample.Currently, experiments in chemistry and biology typically involveevaluating large numbers of samples. Sequencing of nucleic acid samplesis typically time consuming and labor intensive. Therefore, it isdesirable that a large number of samples can be simultaneously analyzed.With large scale projects such as the Human Genome Project, it isdesirable to increase throughput of nucleic acid sequencing.

Electrophoresis is an increasingly common method of performing analysis,e.g. sequencing, of biological substances in order to increasethroughput. Electrophoresis is an electrochemical process in whichmolecules with a net charge migrate in a solution under the influence ofan electric current. Electrophoresis using one or more capillaries whichare illuminated by a laser has proven to be useful in analyzingbiological substances. Existing systems are typically not well-adaptedfor imaging large numbers of samples with a small focal ratio and highlight collecting ability. Therefore, there is a need for an apparatusand method that maintains a substantially uniform image quality over alarge field of view. Preferably, such an apparatus is compact, simple,and reduces focusing problems.

SUMMARY OF THE INVENTION

The advantages and purposes of the invention will be set forth in partin the description which follows, and in part will be obvious from thedescription, or may be learned by practice of the invention. Theadvantages and purposes of the invention will be realized and attainedby the elements and combinations particularly pointed out in theappended claims.

To attain the advantages and in accordance with the purposes of theinvention, as embodied and broadly described herein, the inventionincludes an optical system for analyzing light from a plurality ofsamples. The optical system includes a plurality of sample holders, acollection lens, a reimaging lens, and a light dispersing elementlocated between the collection lens and the reimaging lens. Thecollection lens is configured to receive and substantially collimatelight from the samples. The light dispersing element is configured tospectrally disperse the substantially collimated light from thecollection lens. The reimaging lens is configured to receive light fromthe light dispersing element and direct the light onto a light detectiondevice.

In another aspect of the present invention, the invention is directedtowards a system for analyzing light from a sample in a separationsystem. The system includes at least one separation lane, a collectionlens, a reimaging lens, and a light dispersing element located betweenthe collection lens and the reimaging lens. The light source provides anexcitation light to the at least one separation lane. The collectionlens is configured to receive and substantially collimate light emittedfrom the separation lane. The light dispersing element is configured tospectrally disperse substantially collimated light from the collectionlens. The reimaging lens is configured to receive dispersed light fromthe light dispersing element and direct the light onto a light detectiondevice. In certain embodiments, the system may include a light sourceproviding an excitation light to the at least one separation lane. Incertain embodiments, the system may include a plurality of theseparation lanes.

In yet another aspect of the present invention, the invention includesan optical spectrograph for analyzing light from at least one sample.The optical spectrograph includes at least one source of excitationlight for illuminating at least one sample holder, a first lens unit, atransmission grating, a second lens unit, and a light detection devicehaving a plurality of detector elements. The first lens unit has atleast one lens and is configured to receive and substantially collimatelight from the sample holder. The excitation light from the source ofillumination does not pass through the first lens unit prior toilluminating the at least one sample holder. The transmission grating isconfigured to spectrally disperse substantially collimated light fromthe first lens unit. The second lens unit has at least one lens and isconfigured to receive light from the transmission grating and direct thelight onto the light detection device.

In a further aspect of the present invention, the invention is directedtoward a method of optically analyzing at least one sample. The methodincludes providing at least one holder having a sample therein,illuminating the sample with an excitation light to generate an emissionlight, and collecting the emission light from the sample with acollection lens. In certain embodiments of the method, the excitationlight does not pass through the collection lens prior to illuminatingthe sample. The emission light is substantially collimated by thecollection lens. The method further includes spectrally dispersing thesubstantially collimated emission light beam with a transmissiongrating, directing the emission light from the transmission grating ontoa light detection device by a reimaging lens, and optically detectingthe spectral characteristics of the emission light. In certainembodiments, a plurality of sample holders are provided.

It is to be understood that both the foregoing general description andthe following detailed description are exemplary and explanatory onlyand are not restrictive of the invention, as claimed.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings, which are incorporated in and constitute apart of this specification, illustrate several embodiments of theinvention and together with the description, serve to explain principlesof the invention. In the drawings,

FIG. 1 is a side view of an optical system for analyzing light accordingto certain embodiments of the present invention;

FIG. 2 is a perspective view of an array of capillaries of a separationsystem which may be used with the present invention;

FIG. 3 is a schematic of an optical system having a single lens;

FIG. 4 is a schematic of an optical system according to the presentinvention having two lenses;

FIGS. 5A and 5B are schematics illustrating the effect of a light sourcebeing positioned at the optical axis and offset perpendicular from theoptical axis, respectively, as the light passes through a collectionlens;

FIGS. 6A and 6B are schematics illustrating the light rays of FIGS. 5Aand 5B, respectively, passing through a reimaging lens onto a lightdetection device;

FIGS. 7A and 7B are schematics illustrating the effect of a light havingdifferent wavelength components, such as blue and red light,respectively, as the light passes through the collection lens;

FIGS. 8A and 8B are schematics illustrating the blue and red componentsof FIGS. 7A and 7B, respectively, passing through the reimaging lensonto a light detection device;

FIG. 9 illustrates incident light being spectrally dispersed by atransmission grating;

FIG. 10 illustrates the channels on an optical detection device such asa CCD, according to certain embodiments of the present invention;

FIGS. 11A-11C illustrate the position of an image relative to thedesired image plane for light having a long wavelength, mediumwavelength, and short wavelength, respectively;

FIG. 12 is a schematic illustrating the effect of chromatic aberrationon focusing in certain embodiments of the present invention;

FIG. 13 is a schematic illustrating the effect of tilting a detectionplane of a CCD to reduce chromatic aberration;

FIG. 14 is a side view of a system for testing the amount of tilting ofthe detection plane of the CCD to bring the image into focus;

FIG. 15 is a perspective view of an optical system according to anembodiment of the present invention;

FIG. 16 is a side view of an optical system, illustrating the curvedfield of focus in certain systems that do not include a correction lens;

FIG. 17 is a side view of an optical system according to embodiments ofthe present invention, with a correction lens.

FIG. 18 is a schematic of an optical system with no vignetting;

FIG. 19 is a schematic of an optical system with vignetting;

FIGS. 20A-20D are front views illustrating the effect of vignetting asthe angle of the incident light becomes more offset;

FIGS. 21A-21D are top views corresponding to FIGS. 20A-20D respectively;

FIG. 22 is a side view of an optical system according to certainembodiments of the present invention with a light source on the opticalaxis so that no vignetting occurs;

FIG. 23 is a side view of the optical system of FIG. 22 with the lightsource displaced from the optical axis so that vignetting occurs;

FIG. 24 illustrates a cat's eye aperture according to certainembodiments of the present invention;

FIGS. 25A-25E are front views illustrating the effect of the cat's eyeaperture of FIG. 24 on the light throughput of an optical system;

FIGS. 26A-26E are top views corresponding to FIGS. 25A-25E respectively;and

FIG. 27 is a schematic of an optical system according to an embodimentof the present invention, with a hemispherical element;

FIG. 28 is a side view of an embodiment of the optical system of FIG.27; and

FIG. 29 is a schematic of another optical system with a hemisphericalelement.

DESCRIPTION OF PREFERRED EMBODIMENTS

Reference will now be made in detail to several preferred embodiments ofthe invention, examples of which are illustrated in the accompanyingdrawings. Wherever possible, the same reference numbers will be usedthroughout the drawings to refer to the same or like parts.

According to certain embodiments, the present invention provides anoptical system for analyzing light from a plurality of samples.According to certain embodiments of the invention, the optical systemgenerally includes a plurality of sample holders, a collection lensconfigured to receive and substantially collimate light from the sampleholders, a light dispersing element such as a transmission gratingconfigured to spectrally disperse the substantially collimated lightfrom the collection lens, and a reimaging lens configured to receive thelight from the light dispersing element and direct the light onto alight detection device. Preferably, the sample holders are locatedsubstantially at the image plane of the collection lens. In certainembodiments, the optical system is used in a separation system having aplurality of separation lanes.

The present invention further provides methods of optically analyzing atleast one sample in a spectrograph. The method includes providing atleast one sample holder having a sample therein, illuminating the samplewith an excitation light to generate an emission light, and collectingthe emission light from the sample with a collection lens. In certainpreferred methods, the excitation light does not pass through thecollection lens prior to illuminating the sample. The emission light issubstantially collimated by the collection lens. The method furtherincludes spectrally dispersing the substantially collimated emissionlight with a light dispersing element, such as a transmission grating,directing the emission light from the light dispersing element onto alight detection device by a reimaging lens, and optically detecting thespectral characteristics of the emission light.

FIG. 1 shows an example of an optical system for analyzing light from asample according to certain embodiments of the present invention. Asembodied herein and shown in FIG. 1, optical system 10 includes at leastone sample holder, with a sample contained therein. The sample ispreferably located at or near the object plane 12. The object plane is aplane at the focal distance from the collection lens 14. The opticalsystem further includes a collection lens 14, a light dispersing element16, a reimaging lens 18, and an optical detection device 20. In theexample shown in FIG. 1, the optical system further includes anexcitation light blocking filter 22 positioned between the collectionlens 14 and the light dispersing element 16.

In accordance with the present invention, the optical system includes atleast one sample holder for containing a sample. The present inventionwill be described herein for use with a plurality of sample holders.However, the present invention is also suitable for use with a singlesample holder. In certain preferred embodiments, the sample holderscomprise a plurality of separation lanes, such as electrophoresiscapillaries, located within the object plane of the collection lens. Aseparation lane is a path along which migrating sample components areseparated, for example, using electrophoresis, chromatography,sedimentation, or other separation processes. FIG. 2 shows an example ofa capillary arrangement suitable for use with the optical system ofcertain embodiments of the present invention. This capillary arrangementis known in the art, and is used for purposes of example only. Thecapillary arrangement shown in FIG. 2 is described in greater detail inU.S. Pat. No. 5,498,324 to Yeung et al., the disclosure of which ishereby incorporated in its entirety herein for any purpose. Othersimilar capillary arrangements are described in U.S. Pat. No. 5,938,908to Anazawa et al., the disclosure of which is also hereby incorporatedin its entirety herein for any purpose.

As embodied herein and shown in FIG. 2, the capillary system 200 isparticularly suited for capillary electrophoresis. The capillary system200 includes an array of capillaries 202 with optical fibers 204inserted into an outflow end of the capillaries 202. In certainembodiments, a laser is directed at the ends of the optical fibers.Spacing capillaries 206 with black coating are placed between each ofthe capillaries 202 used for separation. Spacing capillaries 206 act asspacers to prevent crosstalk between separation capillaries 202. Thiscapillary system is described in greater detail in U.S. Pat. No.5,498,324 to Yeung et al. In certain capillary arrangement embodiments,sixteen capillaries are provided. This number may be varied to rangefrom one to several hundred, depending on the field of view and size andquality of the optical system.

Other types of sample holders could be used including oligonucleotidearrays as described for example in U.S. Pat. No. 5,445,934 to Fodor etal., and microfluidic devices as described for example in WO97/36681 toWoudenberg et al., the disclosures of which are both hereby incorporatedby reference in their entirety herein for any purpose. Other types ofsample holders suitable for use with the present invention, include, butare not limited to, wells, slides, test tubes or any other sampleholding device able to confine a sample to a known location. Asgenerically shown in FIG. 2, an optical system 10 of the presentinvention may be positioned perpendicular to the capillary array.

Certain embodiments of the present invention also include an excitationsource such as a laser for generating an excitation light to illuminatethe sample (or samples) in the sample holder (or sample holders). One orseveral excitation sources may be provided. In certain particularembodiments using capillary tubes for holding the samples, excitation isprovided to the sample by an Argon ion laser as discussed generallyabove, and in greater detail in U.S. Pat. No. 5,498,324 to Yeung et al.Other types of conventional excitation sources may also be used, such asan arc lamp (e.g., mercury/xenon lamp, mercury vapor lamp), xenon lamp,tungsten/halogen lamp, deuterium lamp, light emitting diode (LED), orhigh-intensity discharge (HID) lamp. The excitation source is typicallyselected to emit excitation light at one or several wavelengths orwavelength ranges absorbed by the sample or samples. In one specificexample, lasers having a wavelength of 488 and 514 nm are used.

When the sample is illuminated by a laser or other excitation source,the sample acts as a light source and emits an emission light. Incertain particular embodiments, two lasers are used. The provision of alaser or excitation source on opposite sides of the capillaries incertain embodiments helps to provide a more uniform intensity across arow of samples. Some applications of the present invention do notrequire an excitation source in order to illuminate the sample. Forexample, in chemilluminescence and electrochemilluminescence, a sampleemits light without an excitation light source.

Preferably, the sample (or samples) is located at approximately thefocal distance (at the object plane) from the collection lens 14 so thatthe light emitted from the sample (the emission light) upon being struckwith an excitation light (e.g., laser beam) will be optimally collectedby the collection lens 14. The sample is preferably positioned at ornear the object plane of the collection lens.

The collection lens 14 is configured to receive and substantiallycollimate light from at least one sample holder. The collection lens isa type of lens that is spaced from the sample and that can collectlight. An emission light from the sample is collected by the collectionlens and directed toward a light dispersing element. The collection lens14 may include a single lens or an assembly of multiple lenses, but willbe referred to as a single lens for purposes of illustration. Becausethe collection lens is a collimating lens, light from the sampleholder(s) will be converted into substantially parallel (or collimated)light rays as it passes through the collection lens. With a collimatinglens such as collection lens 14, if a source of light (e.g., capillarytube) is centered on the optical axis 15 of the collection lens at theobject plane 12, the emission light from the sample will be collimatedalong the optical axis 15 of the collection lens 14, as shown in FIG.5A.

If the same point source of light is now moved to the side of theoptical axis 15 along the object plane 12, as shown in FIG. 5B, theemission light will be collimated but at an angle θ to the optical axis15 of the collection lens 14. Each point on the collection lens' objectplane 12 (within the field of view of the lens) maps to a unique angle θas light emitted from the point passes through the collection lens.Therefore, in the system of the present invention, a plurality ofspaced-apart sample holders (e.g., capillary tubes) may be imaged by thesame collection lens. A collimating lens such as the collection lens 14functions to convert spatial information into angular information.

The use of a collimating lens such as collection lens 14 provides asubstantially collimated region 26 as shown in FIG. 4. The light issubstantially collimated in the region between the collection lens 14and reimaging lens 18. The collimated region is particularly well suitedfor the insertion of a variety of optical devices (e.g., apertures,interference filters, etc.). An optical lens system with a plurality oflenses (e.g., a relay lens system) can provide advantages over a singlelens system 28 such as shown in FIG. 3. In a single lens system, onelens 30 both collects light from an object plane 12 and refocuses thelight to an image plane 20. The single lens system cannot provide acollimated region for the insertion of optical devices, a disadvantagecompared to the present invention.

A lens that may be used as a collection lens in the present inventioninclude, but are not limited to, any positive lens, i.e., a lens thatbrings collimated light to a focus. These positive lenses may include,for example, a still camera lens, a CCD video camera lens, a microscopeobjective, or an achromatic lens.

In accordance with the present invention, a light dispersing element maybe provided, along with other optical devices, in the substantiallycollimated region 26 between the collection lens 14 and reimaging lens18. As discussed above, the collection lens substantially collimates thelight and directs it to the light dispersing element. A light dispersingelement can be any element that spectrally separates incoming light intoits spectral components. For example, incoming light can be deflected atan angle roughly proportional to the wavelength of the light. Thus,different wavelengths are separated.

For the sake of illustration only, the light dispersing elementdiscussed will be a transmission grating. In a transmission grating, thelight rays that strike the grating surface are transmitted through thegrating. In general, rays of light that strike a transmission gratingsurface deflect at an angle roughly proportional to the wavelength ofthe light. The transmission grating can be of several types, such as aflat blazed grating. Blazed gratings have a triangular, saw-tooth shape.The shape of each groove functions like a prism to refract the light.Typically, a grating will have hundreds or thousands of grooves per mm.In certain particular embodiments, the grating groove density may rangefrom about 100 grooves/mm to about 1,200 grooves/mm. This range is forpurposes of example only, as larger and smaller groove densities mayalso be used with the present invention.

In a transmission grating, the rays of light pass through the gratingand are spread spectrally as is known in the art. FIG. 9 illustrates anincident light 40 striking a transmission grating and being dispersedspectrally into a plurality of distinct light rays 42, 44, 46, 48, eachcorresponding to a particular wavelength component of incident light 40.

This concept is further illustrated in FIGS. 7A and 7B. FIGS. 7A and 7Bshow light being emitted from a point on the object plane at the opticalaxis 15 of the collection lens 14. The emission light is collimated bythe collection lens 14 and strikes the transmission grating 16. When thecollimated light strikes the transmission grating, the light isdispersed spectrally. With spectral dispersion, a first light ray of afirst wavelength will be deflected at a different angle with respect tothe optical axis than a second light ray of a second wavelength. Thiseffect is illustrated in FIGS. 7A and 7B, where the components of thelight having a wavelength corresponding to blue are deflected at adifferent angle than the components of the light having a wavelengthcorresponding to red. The blue rays will remain collimated, as will thered rays, but at different angles with respect to the optical axis ofthe collection lens and with respect to each other.

The light dispersing element spreads the light spectrally in a directionsubstantially perpendicular to the spectral channels on the lightdetection device. This configuration creates a two-dimensional image onthe light detection device after the light passes through the reimaginglens 18; in the first direction the light is spectrally dispersed, in asecond direction the light is spatially resolved/separated. The lightforms a plurality of images corresponding to channels on the lightdetection device so that the light from each channel may be analyzed forits spectral components.

A variety of other types of light dispersing elements, such asreflection gratings or prisms may be used with the present invention,although a transmission grating is preferred. The basic principles ofthe present invention are applicable with a variety of other types oflight dispersing elements such as prisms, grating prisms (“grisms”), andone or more dichroic filters.

The light dispersing element disperses the collimated light and directsthe light toward a reimaging lens. The reimaging lens 18 receives thespectrally dispersed, but still collimated, light from the lightdispersing element and directs it to the light detection device. Thereimaging lens 18, also known as a focusing lens in certain embodiments,takes a collimated light and directs it at or near a point on the imageplane of the reimaging lens. In certain embodiments, the reimaging lens18 is optically identical to the collection lens 14, except that thelens are reversed in direction so that they face each other. The imageformed by the reimaging lens on the light detection device may be at a1:1 ratio with the sample being analyzed, or it may be magnified ordemagnified.

As illustrated in FIGS. 6A and 6B, the reimaging lens 18 receives thesubstantially collimated light from the collection lens, and directs thelight toward an image plane 20. In a manner similar to the collectionlens, if the incident collimated light is centered along the opticalaxis 19 of the reimaging lens 18, the light will be focused on the imageplane 20 at the optical axis 19 of the reimaging lens, as shown in FIG.6A. However, if the incident collimated light is angled relative to theoptical axis 19 of the reimaging lens 18 (such as shown in FIG. 5B), thelight will be focused on or near the image plane offset from the opticalaxis 19 of the reimaging lens, as shown in FIG. 6B. The angle of thecollimated light relative to the optical axis 19 of the reimaging lensdetermines a unique location on the image plane 20 of the reimaging lens(within the field of view). The angular displacement of the light fromthe collection lens is converted into a spatial displacement on thelight detection device (e.g., CCD) by the reimaging lens. In thismanner, the optical system can focus light from a plurality of lightsources (e.g., a plurality of illuminated capillary tubes).

As described in relation to FIGS. 7A and 7B, light from a given sampletypically includes components having different wavelengths. As shown forexample in FIGS. 8A and 8B, these different components (blue and red inthe example shown) will remain collimated after passing through thelight dispersing element. As shown in FIG. 8A, when the blue collimatedrays pass through the reimaging lens, the blue rays will be focused ontoa first location on the image plane 20. As shown in FIG. 8B, the redcollimated rays also pass through the reimaging lens and are focused ona second location on the image plane 20. The second location isdisplaced from the first location. This effect occurs for each of thedifferent wavelengths of light rays, so that a spectrum is created foreach individual sample (e.g., capillary tube). The spectrum for eachcapillary tube (or other type of sample holder) that is formed on thelight detecting device is referred to as a channel. The number ofchannels corresponds to the number of capillary tubes in the field ofview.

Preferably, in certain embodiments, the image plane of the reimaginglens is located coplanar with the light detection device 20 foroptimized detection. A light detection device analyzes light from asample for its spectral components. In certain embodiments, the lightdetection device comprises a multi-element photodetector. As usedherein, the term means a detector having a plurality of addressabledetector elements. Exemplary multi-element photodetectors may include,for example, charge-coupled devices (CCDs), diode arrays,photo-multiplier tube arrays and charge-injection devices (CIDs). A CCDis typical if a plurality of samples are being simultaneously analyzedbecause it can provide an area for a plurality of channels (e.g., onechannel for each sample). The light dispersing element, specifically atransmission grating in certain embodiments, spectrally spreads thelight from each capillary tube so that it can be spectrally analyzed bya CCD. However, if only one sample or capillary tube is used, it may bedesirable to use a single photosensor.

As previously discussed, if a plurality of samples are simultaneouslyanalyzed, a corresponding number of channels will be formed on the lightdetecting device. In certain embodiments, these channels will be formedas parallel channels. An optical detection device such as a CCD has anarray of detection units, or pixels, arranged on a planar surface. A CCDwill typically have a large number of pixels, usually several hundred ineach of the two axis. Each pixel will map to a specific channel orportion thereof and specific color (wavelength) of light from a samplein a specific capillary.

In certain embodiments, the CCD has two axes: a spatial axis and aspectral axis. This is shown in FIG. 10 which illustrates an examplewith five channels 50 on the CCD. Each channel spaced along the spatialaxis (x-axis) corresponds to an individual capillary tube. For example,if sixteen capillary tubes are used, there will be sixteen channels onthe CCD. In certain embodiments, it is preferred to analyze the centerof the sample to get the most accurate results, therefore only thepixels at or adjacent the center of each channel will be analyzed. Incertain embodiments of the present invention, the capillary tubes havean inner diameter of approximately 50 μm, which corresponds to about twoor three pixels. The spectral axis (y-axis) contains spectralinformation (e.g., wavelength vs. intensity information) for each of thecapillary tubes. The axes could also be reversed if desired.

The optical system may further include one or more blocking filters toprevent significant amounts of excitation light or other backgroundlight (e.g., Raman light, ambient light, etc.) from reaching the lightdetecting device. In certain embodiments, in order to block scatteredexcitation light from causing noise (e.g., optical shot noise) in thesystem, one or more excitation blocking filters, such as long-passfilters, may be provided in the emission optical path. Excitationblocking filters prevent certain types of excitation light from enteringthe emission optical path of the optical system. FIG. 1 shows anexcitation blocking filter 22 positioned between the collection lens 14and the transmission grating.

Several types of filters such as interference filters and colored glassfilters may be used as excitation blocking filters in the opticalsystem. There are several types of interference filters such as notch,long-pass, and band-pass filters. There are also several types ofcolored glass filters such as long-pass and band-pass filtersInterference filters may be configured to significantly blockwavelengths below a predetermined threshold from passing to thereimaging lens. Excitation blocking filters may also be placed at avariety of other positions, such as prior to the collection lens (e.g.,between the sample and the collection lens). The collimated region isparticularly suited for interference filters, because interferencefilters typically operate most efficiently when the incident lightstriking the filter is perpendicular to the surface of the filter (andthe light is collimated). An interference filter in the collimatedregion will preferably be positioned between the collection lens and thetransmission grating. One may use more than one excitation blockingfilter at different positions, or may use one excitation blocking filterat any of the possible positions.

The present invention may also be particularly configured to reducechromatic aberration. Chromatic aberration is a variation in focallength (and focus quality) with wavelength. Chromatic aberration may beparticularly troublesome with wavelengths in the non-visible range, suchas infra-red or near infra-red wavelengths. FIGS. 11A-10C illustrate theeffect of wavelength on the extent of chromatic aberration asrepresented by the position of the focal point 23 relative to themid-wavelength image plane 20 of reimaging lens 18. FIG. 11Ademonstrates that for a longer wavelength, the position of focal point23 is beyond the mid-wavelength image plane 20. FIG. 11B demonstratesthat for a middle, optimum wavelength, the position of focal point 23occurs at the mid-wavelength image plane 20. FIG. 11C demonstrates thatfor a short wavelength, the position of focal point occurs before themid-wavelength image plane 20.

FIG. 12 illustrates a possible effect of chromatic aberration on theperformance of the optical system. As shown in FIG. 12, the light raysin the center of the image plane (at the optical axis of the reimaginglens) will be focused, while those at the edges will be out of focus. Itis desirable to minimize chromatic aberration (i.e., have the light raysfocused on the light detection device at both the center and the edgesof the image plane) in order to increase the quality of the imageobtained.

According to certain embodiments of the present invention, tilting theplane of the light detection device 21 (or other elements along theoptical path) can assist in reducing chromatic aberration as describedin FIG. 12. As shown in FIG. 13, tilting the planar surface 21 of thelight detecting device (CCD) by a slight angle with respect to theoptical axis of the collection lens compensates for chromaticaberration, thereby improving the quality of the image on the CCD over awider spectrum of the light wavelength. Other adjustments besidestilting the planar surface of the light detection device may also beused in order to minimize the effect of chromatic aberration on thequality of the image. For example, one or both of the collection lensand transmission grating may be tilted with respect to the optical axisof the collection lens. Moreover, any of the filters may be also betilted with respect to the optical axis of the collection lens. Incertain embodiments, it is desirable to be able to adjust all of theoptical elements in order to bring an image into sharper focus.

The tilting of the light detection device also assists in reducing theamount of stray light that is reflected off of the light detectiondevice back into the system. Stray light may reduce the desired signalto noise values or cause channel to channel cross-contamination. Anothermethod of reducing stray light, besides tilting the light detectiondevice, is to provide anti-reflective coating on the light detectiondevice. The anti-reflective coating may be provided on the window of thelight detection device, or on the planar surface of the light detectiondevice itself. It may also be desirable to provide an anti-reflectivecoating on any and all of the elements that the light passes through.

In accordance with certain embodiments of the present invention, adevice and technique for determining the optimal angle of tilt of aplanar light detection device is provided. In this technique, thedetection device, e.g., CCD, may be tilted at various angles and theresulting images examined for focus quality along a spectral axis of thelight detection device. The focus along the edges of the image is thencompared to the focus in the center of the image. As embodied in certainembodiments shown in FIG. 14, the system includes a collection lens 60,transmission grating 62, reimaging lens 64, and charge-coupled device(CCD) 66, which may be identical to the collection lens 14, transmissiongrating 16, reimaging lens 18, and charge-coupled device at image plane20, respectively, of the system previously described for FIGS. 1-13. Inthe testing apparatus, the charge-coupled device 66 is tilted bypivoting about rotational axis 68 shown in FIG. 14. The device formeasuring the effect of the tilting includes a test aperture 70 with alight diffuser, a light source 72 such as a neon bulb, and an x-y stage74. It was discovered that a slight change in the angle of the CCD maysubstantially reduce the amount of chromatic aberration in the opticalsystem.

One particular example of the optical system of the present invention isillustrated in FIG. 15. The system shown in FIG. 15 is by way of exampleonly, and is not meant to be limiting in any manner. As embodied hereinand shown in the example of FIG. 15, the optical system includes acollimating assembly 80 with a collimating lens therein, a gratingassembly 82 with a transmission grating therein, an angle assembly 84, afocusing assembly 86 with a reimaging lens therein, and a CCD assembly88. In this particular example, the collating assembly 80 and focusingassembly 86 include camera lenses, such as Nikon camera lenses with anaperture speed of f/1.4. Camera lens having higher or lower aperturespeeds, such as f/1.2, for example, are also suitable with the presentinvention. In the example shown in FIG. 15, the camera lens each have afocal length of 50 mm.

The grating assembly 82 includes a transmission grating similar totransmission grating 16 previously described. In the example of FIG. 15,the transmission grating has a ruling of 600 grooves/mm. The angleassembly 84 may include optical elements such as one or more correctionlenses, apertures, or gratings. Preferably, the angle assembly 84 isconfigured to be angled to match the amount of diffraction caused by thetransmission grating in the grating assembly 82 at a mid-wavelength ofinterest. The CCD assembly 88 includes a charge coupled device aspreviously described. The CCD has a pixel spacing of 24 □m/pixel and anarray of 512×250 pixels. Moreover, the optical system includes aninterference filter (not shown in FIG. 15) positioned between thecollimating assembly 80 and the object plane of the sample. The opticalsystem also includes a second interference filter (not shown in FIG. 15)positioned between the collimating assembly 80 and the grating assembly82. These two interference filters may be identical. The optical systemof FIG. 15 is for purposes of example only.

In the present invention, the optical components may be mounted in anymatter known in the art. For example, the components may be placed onsupports or other mechanical supporting means and attached to an opticaltable.

In accordance with other embodiments of the present invention, theoptical system may include a correction lens. A correction lens canreduce the curvature of the field of focus at the image plane of a lensoptimized for a larger field of view than required for the presentinvention. As embodied herein and shown in FIG. 17, a correction lens 90may be positioned between the collection lens 14 and the reimaging lens18. The correction lens may be utilized in particular systems in whichcurvature is an issue. In the optical system of FIG. 16, the combinationof camera lenses 14 and 18 typically result in an overcorrected focusfield curvature, such as shown in dashed lines by reference numeral 92.This curved field of focus does not typically cause problems inphotographic applications. However, in an optical system used forspectrography, the area of interest is limited to a small percentage ofthe area of interest typically used in normal photography.

In order to compensate for the overcorrection of the system of FIG. 16,a correction lens 90 may be inserted between the collection lens 14 andthe reimaging lens 18. In certain embodiments, the correction lens is asimple achromatic lens 90 as shown in FIG. 17. The correction lenstypically should be configured to reduce the curvature of the field offocus, resulting in a substantially planar field of focus such as shownby reference numeral 94 in FIG. 17. Moreover, the correction lenstypically should be configured to provide the collimated region aspreviously described. The use of a correction lens may result in amagnification or demagnification of the image, as shown in FIG. 17.

In accordance with other embodiments of the present invention, theoptical system reduces variations in light throughput across the fieldof view, typically referred to as vignetting. Vignetting can be alimiting effect of an optical system where portions of entering lightare not permitted to pass through the optical system because ofstructural obstacles. FIG. 18 illustrates an optical system with a fulllight throughput, with no vignetting. In FIG. 18, all of the light thatenters the first lens 100 is fully transmitted to the second lens 102,without any structural obstacles blocking the light. FIG. 19 illustratesthe effect of vignetting. In FIG. 19, the light source is locatedoff-center from the optical axis of the first lens 100. Therefore, onlya portion of the light is transmitted through the second lens 102 to theimage plane 20. The light throughput of the optical system has droppedbecause of vignetting. With certain embodiments of the presentinvention, it may be desirable to reduce vignetting, particularly ifcapillaries (or other sample holders) are placed near the limits of thefield of view of the collection lens.

FIGS. 20A-20D and 21A-21D illustrate the effect of vignetting on ahypothetical system as the light source becomes more distant from theoptical axis of the system. In this hypothetical system, the system isshown as 110. As shown in FIGS. 20A and 21A, all the incident light 111will pass through the system 110 as exit light 113, when the lightsource is located exactly on the optical axis of the system. FIGS. 20Band 21B illustrate the light being at a slight angle to the optical axisof the system. As can be seen in FIGS. 20B and 20B, a small amount ofthe outer fringes of the incident light 111 will be clipped by thesystem. A large percentage of the incident light 111 exits as exit light113 in the arrangement of FIGS. 20B and 21B, resulting in a small amountof vignetting. FIGS. 20C and 21C show an increase in the angle of theincident light relative to the system, and FIGS. 20D and 21D show aneven greater increase in the angle of the incident light relative to thesystem. The amount of vignetting increases as the angle of the incidentlight 111 relative to the system optical axis increases. As is alsoclear from these drawings, the amount of light exiting the system (lightthroughput) decreases. In certain embodiments, it is desirable to avoida decrease in light throughput in an optical spectrograph system.

FIGS. 22 and 23 illustrate the possible effect of vignetting on anoptical system with lenses similar to those previously discussed. FIG.22 shows the light source being positioned on the optical axis of thecollection lens 14. As seen in FIG. 22, emission light from the lightsource passes through the optical system to the image plane 20 withoutinterference. In FIG. 23, the light source is positioned at the objectplane, but spaced perpendicularly from the optical axis. As shown inFIG. 23, a portion of the light is blocked by the internal structure ofthe optical system. Therefore, a portion of the initial light is lost,and the light throughput decreases. The optical system of certainembodiments of the present invention is particularly configured toreduce vignetting by placing the reimaging lens 18 as close to thecollection lens 14 as possible. This helps to reduce the amount of lightthat is blocked from passing through the system, thereby reducingvignetting.

Certain embodiments of the present invention are directed toward adevice and method for reducing variations in light throughput in anoptical system, which may be a result of excessive vignetting. Asembodied herein and shown in FIG. 24, the optical system of certainembodiments of the present invention may include a light blockingaperture 112 positioned between the collection lens 14 and the reimaginglens 18. The light blocking aperture 112 is preferably positioned atapproximately the position of filter 22 in FIG. 1, between thecollection lens 14 and the light dispersing element 16. Preferably, theaperture 112 is configured to have a particular geometric shape such asthe “cat's eye” or football shape shown in FIG. 24. The cat's eye isgeometrically described as being two arcs joined at a top and bottom,with the width being the smallest at the top and bottom, and greatest atthe middle. The “cat's eye” aperture is generally in the shape of anAmerican football, as is shown in FIG. 24. In other embodiments, theaperture could be in other shapes such as circular.

In certain embodiments, the cat's eye aperture 112 is positioned exactlymidway between the collection lens 14 and the reimaging lens 18. Incertain particular embodiments, the cat's eye aperture 112 is positionedat the location where the excitation blocking filter 22 is shown inFIG. 1. The aperture blocks the light so that the amount of light thatpasses through is always substantially equal to the throughput of theworst off-axis rays striking the collection lens. This is illustrated inFIGS. 25 and 26. The cat's eye aperture allows substantially uniformlight throughput even when the angle of the input rays varysubstantially. The overall throughput of a system with this aperturewill be lower compared to a system without an aperture, however this isacceptable in certain embodiments in view of the specifically statedadvantages.

FIGS. 25A-25E and 26A-26E demonstrate how, in a system with a cat's eyeaperture, a substantially uniform light throughput occurs even thoughthe angle of the incident light relative to the system varies. FIG. 25illustrates the view looking down the optical system with the cat's eyeaperture. FIG. 26 illustrates a top view of the system with a cat's eyeaperture 112 positioned approximately midway through the system. FIGS.25A and 26A illustrate the incident light 111 at its most off-axis(e.g., at its greatest angle to the optical axis of the system) positionto the left of the system. A light throughput of a predetermined amountis permitted to pass through the optical system. The exiting light isrepresented as reference number 113. FIGS. 25B and 26B illustrate thelight throughput when the incident light is at a lower angle relative tothe optical axis of the system. As seen in the FIGS. 25B and 26B, theshape of the light that passes through the cat's eye aperture (andoptical system) is identical to the shape of the light that passedthrough the cat's eye aperture in FIGS. 25A and 26A. The amount of lightthroughput therefore does not vary with the angle of the incident light.

FIGS. 25C and 26C show the light throughput when the incident light iscentered along the optical axis of the system. The shape of the lightand the amount of throughput (shown as exit light 113) remains the sameas for the previous positions. The same uniform light throughput isfound at the positions shown in FIGS. 25D and 26D, and FIGS. 25E and26E.

Uniform light throughput is an advantageous feature in an optical systemfor a spectrograph. With uniform light throughput for every lightdetection device channel (or every sample holder being analyzed), thedynamic range of the system may be increased. This is particularly trueif the integration time of the system is adjusted to approach the fullwell charge capacity of the CCD during operation. Another advantage ofthe cat's eye aperture is that it reduces the f/number of the system agreater amount in the spatial dimension than in the spectral dimension.This increases the image quality in the spatial direction. Spatial imagequality may be particularly important to optimize in order to reducelight from one channel from bleeding over into an adjacent channel.

In accordance with certain embodiments of the present invention, theoptical system may include a first substantially hemispherical opticalelement positioned between the collection lens and the object plane ofthe collection lens. A hemispherical element similar to thehemispherical element of the present invention is described in ABI PRISMDNA Analyzer Service Manual, Revision A., the contents of which arehereby incorporated herein for any purpose. In certain embodiments, theoptical system also includes a second substantially hemisphericaloptical element positioned between the reimaging lens and the imageplane of the reimaging lens. The hemispherical element or elementsassists in increasing the light collection efficiency of the opticalsystem. The hemispherical element or elements preferably increases theaperture speed (f/number) of the system without degrading the imagequality.

One embodiment of the optical system with at least one hemisphericalelement as shown in FIG. 27. As embodied herein and shown in FIG. 27,the optical system 300 includes a first hemispherical element 302 and asecond hemispherical element 304. In certain embodiments, the firsthemispherical element 302 includes a substantially hemispherical outersurface 304 and a flat surface 306 as shown schematically in FIG. 27.The hemispherical outer surface 304 has a radius of curvature R and acenter of curvature at point C. Preferably, the center C of curvature ofthe hemispherical outer surface 304 is positioned at the light source(e.g., at the intersection of the optical axis 308 of the collectionlens 14 and the object plane 12 of the collection lens 14). Alternately,the center of curvature C may be slightly spaced from the image plane 12as shown in FIG. 27. In certain embodiments, it is desired to have theflat surface 306 be as close to the sample as possible. If a slide orother flat surface is used for the sample holder, the flat surface ofthe hemispherical element may be pressed against the surface of thesample holder. In other embodiments, the flat surface of thehemispherical element may be spaced from the top of the sample holder bya minimal amount. It is preferable for the radius of curvature of thehemispherical element to be as large as possible based on the spacingbetween the object plane 12 and the collection lens 14. As seen in FIG.27, the radius of curvature R of the hemispherical element 302 is sizedto be almost as large as the spacing between the collection lens 14 andthe object plane 12.

The optical system 300 may further include a second hemisphericalelement 322. The second hemispherical element 322 includes asubstantially hemispherical outer surface 324 and a flat surface 326 asshown schematically in FIG. 27. The hemispherical outer surface 324 hasa radius of curvature R′ and a center of curvature at point C′.Preferably, the center C′ of curvature of the hemispherical outersurface 324 is positioned at the light detection surface (e.g., at theintersection of the optical axis 308 and the image plane of thereimaging lens 18.) Alternately, the center of curvature C may beslightly spaced from the image plane 12 as shown in FIG. 27. The secondhemispherical element 322 functions similarly to the first hemisphericalelement 302.

The hemispherical elements are constructed of any optically transparentmaterial such as glass and plastic. Preferably, these materials have ahigh index of refraction. Materials that are particularly suited for thehemispherical elements, include, for example, glasses such as flintglass and sapphire, and polymers such as polycarbonates. In certainembodiments, the index of refraction is uniform throughout thehemispherical element.

The hemispherical element 302 allows light rays to proceed from a pointon the sample (positioned at the intersection of the optical axis 308and the object plane 12), to exit the hemispherical element in adirection normal to the hemispherical outer surface 304 (i.e., the lightrays travel from the center C of the radius of curvature to thehemispherical outer surface 304). As shown in FIG. 27, the angle of agiven light ray exiting the hemispherical element to the collection lens14 does not vary when it leaves the hemispherical element. Bymaintaining a straight path for the light rays (between the sample andthe collection lens), aberration is minimized, so that focus is notaffected. For samples that are spaced from the optical axis, the lightrays passing through the hemispherical element and leaving the outerhemispherical surface 304 will be substantially normal, withoutsignificant aberration. The hemispherical element increases thepercentage of the light from the sample that is available to reach thecollection lens 14, thereby increasing the light throughput of thesystem.

The geometry of the hemispherical element 302 is shown schematically asa single element in FIG. 27. The hemispherical element may also becomposed of several different elements as shown in FIG. 28. In FIG. 28,the hemispherical element 302 is composed of several different elements,such as a curved member 330 and rectangular optical elements 332, 334,and 336. In the embodiment of FIG. 28, the rectangular element 336 maybe a slide or other sample holder. The samples 338 are preferablypositioned at the object plane from the collection lens 14. Each of theelements 330, 332, 334, and 336 preferably have a uniform index ofrefraction.

As shown in FIG. 28, the optical system further includes a blockingfilter 22 as discussed previously. Alternately, an aperture may bepositioned at the location of the blocking filter 22. The optical systemfurther includes a light dispersing element 16, such as a transmissiongrating, and a reimaging lens 18. The second hemispherical element 322may also include a plurality of elements, such as curved member 350 andrectangular optical element 352. The second hemispherical element issized so that the radius of curvature is slightly less than the spaceprovided between the reimaging lens 18 and the light detection device ator near object plane 20. If a light detection window 354 is provided asshown in FIG. 28, the second hemispherical element 322 will be designedto fill substantially all of the space between the reimaging lens 18 andthe light detection window 354 of the light detection device.

According to other embodiments of the present invention, thehemispherical element may be configured so that it slightly magnifiesthe image of the sample. This is shown for example in optical system 360of FIG. 29, where a hemispherical element 362 has a center of curvatureC spaced from the object plane 12. The radius of curvature R in FIG. 29is smaller than the radius of curvature described in FIG. 27. As shownin FIG. 29, the hemispherical element 362 may include an additionaloptical element 364 so that a significant gap does not exist between aflat surface of the hemispherical element and the object plane 12. Thesecond hemispherical element 372 may have a radius of curvature R′ lessthan the distance between the reimaging lens 18 and the image plane 20,for purposes similar to those described for the first hemisphericalelement 362. The second hemispherical element may also include one ormore additional optical elements 374.

Various other embodiments exist for use with a hemispherical element.For example, it may be desirable to have some of the elements of ahemispherical element made from elements with different indexes ofrefraction. This may be particular suitable in optical systems in whichaberration is a problem. In such a design, an element having a higher orlower index of refraction than the other elements may be inserted at anysuitable location inside the hemispherical element. In otherembodiments, curved surfaces may be provided inside of the hemisphericalelement.

The hemispherical element may be configured to be other shapes thanperfectly hemispherical. For example, the hemispherical element may beslightly non-hemispherical or aspheric. Other suitable alternategeometries may also be acceptable with the present invention.

Methods of optically analyzing light from a sample are apparent from thedescription of the various embodiments of the optical system above. Themethods include providing at least one sample holder having a sampletherein. One example of suitable sample holders are the separationcapillaries 202 shown in FIG. 2. As previously discussed, a variety ofother types of sample holders are also acceptable. The method furtherincludes illuminating the sample with an excitation light to generate anemission light. The sample, or plurality of samples, may be illuminatedby a variety of different excitation sources such as lasers, aspreviously discussed. One or several of such excitation sources may beprovided. The samples in the sample holders are caused to fluoresce bythe excitation source so that they emit an emission light.

The emitted light from the sample holders is then collected by acollection lens (or first lens unit) that substantially collimates thelight. The collimated light is directed toward a light dispersingelement, such as a transmission grating, which spectrally disperses thesubstantially collimated light. Each of the various wavelengths isdispersed at a distinct angle by the light dispersing element. Thedispersed and collimated light from the light dispersing element is thendirected onto a light detection device by a reimaging lens (or secondlens unit). The light detection device is preferably locatedsubstantially at the image plane of the reimaging lens. The lightdetection device, for example, a two-dimensional multi-element planardetector with a plurality of detection elements, detects the spectralcharacteristics of the emission light. The spectral characteristics maythen be analyzed by any means such as a computer.

The method may also comprise other procedures such as blocking asignificant portion of light having a wavelength lower than apredetermined wavelength using an interference filter. Moreover, tocompensate for possible chromatic aberration, the light detection deviceor other elements may be tilted with respect to the optical axis of thecollection lens so that light is focused on a plane of the lightdetection device. Alternately, or in addition to tilting the lightdetection device, at least one of the reimaging lens 18 and transmissiongrating 16 may be tilted with respect to the optical axis of thecollection lens so that light is focused on a plane of the lightdetection device. The method may also include selectively blocking thelight between the collection lens and the reimaging lens with a footballshaped aperture. The method may further include positioning a correctionlens between the collection lens and the reimaging lens to reducecurvature of the image on the light detection device. Other methodssuitable with the optical system described above may also be used.

It will be apparent to those skilled in the art that variousmodifications and variations can be made in the optical systems, methodsof optically analyzing light from a sample, use of the apparatus of thepresent invention, and in construction of this apparatus, withoutdeparting from the scope or spirit of the invention.

Other embodiments of the invention will be apparent to those skilled inthe art from consideration of the specification and practice of theinvention disclosed herein. It is intended that the specification andexamples be considered as exemplary only, with a true scope and spiritof the invention being indicated by the following claims. All documentscited herein are incorporated by reference for any purpose.

1. An optical system for analyzing light from a plurality of samples,comprising: a plurality of capillaries, wherein the capillaries containthe plurality of samples and each sample in each capillary emits lightin multiple colors during electrophoresis separation; a collection lensconfigured to receive and substantially collimate the light from theplurality of capillaries; a transmission grating configured tospectrally disperse the substantially collimated light from thecollection lens; and a reimaging lens configured to receive thespectrally dispersed light from the transmission grating and direct thelight onto a light detection device, wherein the system is configured tocompensate for chromatic aberration in the spectrally dispersed light,the compensation comprising an optical axis of the reimaging lens beingnon-perpendicular with a surface of the light detection device.
 2. Thesystem of claim 1, wherein the system further comprises the lightdetection device, and wherein said light detection device is atwo-dimensional, multi-element photodetector having respectivelyperpendicular spatial and spectral detection axes.
 3. The system ofclaim 2, wherein light from the plurality of capillaries is spatiallydispersed along the spatial detection axis of the photodetector, suchthat light from each capillary can be detected.
 4. The system of claim3, wherein light from each capillary is spectrally dispersed along thespectral detection axis of the photodetector, such that the multiplecolors from each capillary can be detected.
 5. The system of claim 4,wherein the system is configured to independently detect the multiplecolors.
 6. The system of claim 1, further comprising an excitation lightsource for illuminating the plurality of capillaries.
 7. The system ofclaim 6, wherein light from the excitation light source does not passthrough the collection lens prior to illuminating the plurality ofcapillaries.
 8. The system of claim 7, wherein the excitation lightsource is an LED.
 9. The system of claim 1, wherein the system furthercomprises the light detection device, and wherein the light detectiondevice comprises a charge-coupled device (CCD).